Motor and sensory nerves regenerate in the periphery after being axotomized following nerve injuries. This capacity allows some functional recovery, however this is frequently suboptimal being the relative inefficiency of axon regeneration a major problem, particularly for injuries at some distance from muscle. Axotomy induces genetic changes in motoneurons that promote axon growth, yet this is rather slow taking months or years for axons to reach their targets after limb injuries. This compounds with the fact that the regenerative capacity of motoneurons is limited to a short temporal window and that chronic denervation results in muscle atrophy, as well as changes in central circuits, all impairing recovery. Therefore there is renewed interest on mechanisms to promote axon regeneration. One mechanism recently highlighted and intensely studied by one of the P.I.s (Dr. A.W. English) is the effect of activity and exercise in promoting axon regeneration. However, this approach is limited since patients are frequently either with the affected limbs immobilized or in bed rest preventing implementation of adequate exercise programs. We now seek proof for a mechanistic explanation that could be recruited also with passive rehabilitation and/or pharmacology. After axotomy motoneurons increase their excitability and shed excitatory synapses while maintaining inhibitory synapses. In addition, the potassium chloride co-transporter isoform 2 (KCC2) is downregulated changing the nature of inhibitory synapses from hyperpolarizing to depolarizing. The other P.I. in this proposal (Dr. F.J. Alvarez) is an expert in spinal inhibitory interneurons and synapses. Together, both P.I.s hypothesized that after axotomy inhibitory synapses are the main drivers of motoneuron activity and could stimulate axon regeneration. There is a strong scientific premise for this hypothesis: GABA actions promote axon elongation during early development and manipulations that enhanced preservation of inhibitory synapses on axotomized motoneurons correlated with faster functional recovery. To directly test whether inhibitory synaptic activity on axotomized motoneurons promotes axon regeneration we propose in Aim 1 to block inhibitory synapses on axotomized motoneurons using tetanus neurotoxin A and study the effects on motor axon regeneration and muscle reinnervation. In Aim 2 we will use mouse models to genetically define the interneurons targeting the cell body of regenerating motoneurons and that could provide a depolarizing synaptic drive. We will then use genetically-encoded activity modifiers to examine whether their activity influences motor axon regeneration. Resolution of these aims will reveal for the first time whether inhibitory synapse activity is a driving force for axon regeneration in the adult and the spinal neurons that might be responsible. This is of high significance from a translational point of view since it will point to new approaches to enhance ?inhibitory? drive by either pharmacological means or by recruiting key interneurons through various manipulations, like stretching or stimulating the antagonist muscles and nerves.